Project Summary The notion that pathogens acquire antibiotic resistance from members of the human microbiome is gaining traction, although this has yet to be shown directly. Antibiotic resistance and other critical functions for bacterial survival, including mercury resistance, carbohydrate degradation proteins, virulence factors, and catabolic enzymes, are transferred between bacteria through the processes of horizontal gene transfer. Despite the importance of this process for the spread of antibiotic resistance, surprisingly little is known about the scope and dynamics of HGT in natural bacterial settings. Our limited knowledge is due to the inherent difficulties of assaying the flexible portion of bacterial genomes, with most HGT-related studies focusing only on single organisms, single genes or specific components of the mobile gene pool. Comprehensive, high-throughput methods to examine the mobile gene pool are therefore needed to understand the role of HGT in natural communities at a systems-level and to identify interventions that may reduce the overall transfer of antibiotic resistance genes within the human microbiome. My overall vision is to examine the scope and dynamics of HGT in the human gut microbiome, by developing both experimental and in silico methods for detecting HGT. I will be adapt and employ single-cell technologies that induce long-range linkages in the DNA so that mobile genes can be associated with the species in which they reside. Using metagenomic shotgun data, I will develop an assembly method specifically geared towards mobile genetic elements focused on retaining the heterogeneity in genomic contexts in which mobile genes reside. I will also employ quantitative models to assign bacterial hosts to specific mobile genes. This suite of tools should provide comprehensive information on the origin, gene content and penetrance of mobile genetic elements to capture what is currently being transferred. Since the frequency of HGT in the human microbiome is unknown, I will establish baseline dynamics and further test the hypothesis that specific perturbations to our microbiomes induce horizontal gene transfer. DNA damage and cellular stress pathways increase bacterial competence and the mobilization of transferrable genetic elements. Using a combination of mouse models and studies with human subjects, I will test whether our behaviors that induce DNA damage and cellular stress, namely antibiotic use, infection, and fecal microbiota transplant, result in an increase of horizontal gene transfer and ultimately in the spread of antibiotic resistance genes.